Technical Field
The present invention relates to the microbiological industry, and specifically to a method for producing L-amino acids by fermentation of a bacterium of the family Enterobacteriaceae that has been modified to disrupt putrescine degradation pathway.
Background Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765 A) and alteration of regulatory regions such as promoter, leader sequence, and/or attenuator, or others known to the person skilled in the art (see, for example, US20060216796 A1 and WO9615246 A1). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to the feedback inhibition by the resulting L-amino acid (see, for example, WO9516042 A1, EP0685555 A1 or U.S. Pat. Nos. 4,346,170 A, 5,661,012 A, and 6,040,160 A).
Another method for enhancing L-amino acids production yields is to attenuate expression of a gene or several genes which are involved in degradation of the target L-amino acid, genes which divert the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of the carbon, nitrogen, and phosphate fluxes, and genes encoding toxins, etc.
The pathways that result in the production of arginine, ornithine, putrescine, and γ-aminobutyric acid (GABA) catabolism are related (Schneider B. L. et al., Arginine catabolism and the arginine succinyltransferase pathway in Escherichia coli, J. Bacteriol., 1998, 180(16):4278-4286). Arginine and ornithine are degraded via the ammonia-producing arginine succinyltransferase (AST) pathway to glutamate by AST enzymes encoded by the astCADBE operon genes. The AST pathway is necessary for arginine degradation during nitrogen-limited growth, and it contributes to the degradation of other amino acids. Alternatively, to the AST pathway, another arginine catabolic pathway via agmatine to putrescine exists, which utilizes arginine decarboxylase (ADC) in the initial reaction (ADC pathway). The ADC pathway is not used to degrade arginine when it is the sole nitrogen source (Schneider B. L. et al., Putrescine catabolism is a metabolic response to several stresses in Escherichia coli, Mol. Microbiol., 2013, 88(3):537-550). Contrary to the arginine degradation pathway, ornithine is directly converted to putrescine by ornithine decarboxylase (ODC) in the ODC pathway.
Putrescine, which can also be referred to as tetramethylenediamine or 1,4-diaminobutane, is degraded via GABA to succinate for use as a carbon and nitrogen source by either the glutamylated putrescine (GP) pathway or the transaminase pathway. The GP pathway, also referred to as Puu pathway, utilizes the enzymes to completely degrade putrescine to succinate. The puuA, puuB, puuC, puuD and puuE genes, along with puuP and puuR genes encoding a putrescine/H+ symporter PuuP and DNA-binding transcriptional repressor PuuR, are organized in the puu divergon. The puu gene cluster was found in Escherichia coli (E. coli) and closely related enterobacteria (Nemoto N. et al., Mechanism for regulation of the putrescine utilization pathway by the transcription factor PuuR in Escherichia coli K-12, J. Bacteriol., 2012, 194(13):3437-3447). It is postulated that E. coli and related enterobacteria may utilize the GP pathway as an adaptation for survival in the mammalian intestine, an environment in which polyamines exist at relatively high concentrations. PatA, PatD, GabT, and GabD constitute the transaminase pathway of putrescine degradation (Schneider B. L. et al., 2013, 88(3):537-550).
Until now, no data has been reported demonstrating the effect of the disruption of putrescine degradation pathway on L-amino acid production by modified bacterial strains of the family Enterobacteriaceae.